TCR recognition of peptide antigens bound and presented by MHC proteins is a cornerstone of cellular immunity. Although structural knowledge of TCR-pMHC interactions has surged over the past decade, there are significant gaps in our understanding of how TCRs engage pMHC and achieve the key immunological phenomena of specificity, cross-reactivity, and MHC restriction. Indeed, recent years have seen controversy regarding the determinants of TCR recognition properties, such as the extent to which TCRs are evolutionarily biased towards engaging MHC proteins and how such bias may be influenced by other factors. Additionally, structural immunologists are focusing increased attention on molecular motion, which has been shown to have complex and poorly understood influences on specificity, cross-reactivity, and potentially even signaling. Beyond informing basic immunology, an improved understanding of how TCRs achieve specificity, cross-react, and signal is crucial as antigenic epitopes and TCRs begin to be used in clinical settings. This competitive renewal proposal describes an ambitious plan to address key questions related to the issues above, with goals of not only uncovering fundamental principles, but also advancing the development of TCR and pMHC-based therapeutics. There are two broadly complementary specific aims. The first will determine how the distribution of TCR-pMHC binding energy is shaped by evolutionary and non-evolutionary forces. A range of experimental approaches will be applied, including double mutant cycle analyses, X-ray crystallography, protein engineering, and structural bioinformatics. The second aim will establish the role and impact of molecular motion in TCR and pMHC recognition. A range of approaches will again be used, including NMR, fluorescence, and computation. Throughout both aims, a number of novel hypotheses will be tested, including our hypothesis that the TCR binding site has evolved a structural and energetic permissiveness that permits the receptor to adjust to the unique chemistry present in each interface. We will also test the hypotheses that peptide modulation of MHC dynamics influences NK receptor binding, and that alterations in TCR dynamics upon binding contribute to T cell signaling.